Abstract

Protein expression in formalin-fixed, paraffin-embedded patient tissue is routinely measured by Immunohistochemistry (IHC). However, IHC has been shown to be subject to variability in sensitivity, specificity and reproducibility, and is generally, at best, considered semi-quantitative. Mass spectrometry (MS) is considered by many to be the criterion standard for protein measurement, offering high sensitivity, specificity, and objective molecular quantification. Here, we seek to show that quantitative immunofluorescence (QIF) with standardization can achieve quantitative results comparable to MS. Epidermal growth factor receptor (EGFR) was measured by quantitative immunofluorescence in 15 cell lines with a wide range of EGFR expression, using different primary antibody concentrations, including the optimal signal-to-noise concentration after quantitative titration. QIF target measurement was then compared to the absolute EGFR concentration measured by Liquid Tissue-selected reaction monitoring mass spectrometry. The best agreement between the two assays was found when the EGFR primary antibody was used at the optimal signal-to-noise concentration, revealing a strong linear regression (R2=0.88). This demonstrates that quantitative optimization of titration by calculation of signal-to-noise ratio allows QIF to be standardized to MS and can therefore be used to assess absolute protein concentration in a linear and reproducible manner.

Conflict of interest statement

Disclosure/Duality of Interest:

In the last 24 months I have been engaged in the following relationships: I am a consultant to Astra Zeneca, Agendia, Bethyl Labs, Biocept, BMS, Cell Signaling Technology, Cernostics, ClearSight, FivePrime, Genoptix/Novartis, Metamark Genetics, Merck, OptraScan, Perkin Elmer, and Ultivue. I have received honoraria for presentations at Genentech/Roche, Cell Signaling Technology, and Ventana. I hold equity in Metamark Genetics. Cepheid, Genoptix, Gilead Sciences, Pierre Fabre, Perkin Elmer and Nantomics fund research in my lab.

Figures

Figure 1

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(A) Titration curve of EGFR…

Figure 1

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(A) Titration curve of EGFR D38B1 primary antibody plotted at five different concentrations.…

Figure 1

(A) Titration curve of EGFR D38B1 primary antibody plotted at five different concentrations. The blue line shows the average QIF scores of the lowest 10% patient cases included in the standardization array, representing the noise. The red line shows the average QIF scores of the highest 10% patient cases included in the standardization array, representing the signal. The green line is the signal-to-noise ratio for each EGFR D38B1 antibody concentration. (B) Reproducibility of EGFR expression quantified by AQUAâ in the optimal EGFR D38B1 concentration measured by AQUA in 15 cell lines with different EGFR expression. Red dots represent EGFR mutant cell lines and blue dots represent EGFR wild type cell lines. (C) AQUA images of FFPE cell lines with different EGFR expression level and mutation status. EGFR (red), cytokeratin (green), DAPI (4′,6-diamidino-2-phenylindole) (blue). Images are representative of two independent experiments.

Figure 2

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(A–E) QIF scores in the…

Figure 2

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(A–E) QIF scores in the 15 cell lines with different EGFR expression as…

Figure 2

(A–E) QIF scores in the 15 cell lines with different EGFR expression as measured by AQUAâ at different EGFR D38B1 primary antibody concentrations covering two orders of magnitude in serial sections. (F) Absolute EGFR concentration measured by LT-SRM-MS in 15 cell lines with different EGFR expression. The QIF scoring and the absolute protein concentration by LT-SRM-MS were performed in cores and sections respectively, coming from the same cell pellets. Red bars represent EGFR mutant cell lines and blue bars represent EGFR wild type cell lines.

Figure 3

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(A–E) Regression charts between absolute…

Figure 3

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(A–E) Regression charts between absolute EGFR concentration measured by LT-SRM-MS and the QIF…

Figure 3

(A–E) Regression charts between absolute EGFR concentration measured by LT-SRM-MS and the QIF scores at different EGFR D38B1 primary antibody concentrations covering two orders of magnitude in 15 cell lines, with A431 included. The QIF scoring and the absolute protein concentration by LT-SRM-MS were performed in cores and sections respectively, coming from the same cell pellets. Red dots represent EGFR mutant cell lines and blue dots represent EGFR wild type cell lines.

Figure 4

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(A–E) Regression charts between absolute…

Figure 4

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(A–E) Regression charts between absolute EGFR concentration measured by LT-SRM-MS and the QIF…

Figure 4

(A–E) Regression charts between absolute EGFR concentration measured by LT-SRM-MS and the QIF scores at different EGFR D38B1 primary antibody concentrations covering two orders of magnitude in 14 cell lines, with A431 not included. The QIF scoring and the absolute protein concentration by LT-SRM-MS were performed in cores and sections respectively, coming from the same cell pellets. Red dots represent EGFR mutant cell lines and blue dots represent EGFR wild type cell lines.

References

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